Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus

Van-Trinh Luu; Hye Yun Moon; Jee Youn Hwang; Bo-Kyu Kang; Hyun Ah Kang

doi:10.1007/s12275-017-7218-5

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Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus: Van-Trinh Luu ¹, Hye Yun Moon ¹, Jee Youn Hwang ², Bo-Kyu Kang ³, Hyun Ah Kang ^1,4; Journal of Microbiology 2017;55(8):655-664.
DOI: https://doi.org/10.1007/s12275-017-7218-5
Published online: July 28, 2017

¹Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 06974, Republic of Korea, ²Aquatic Disease Control Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea, ³Green Cross Veterinary Products Co. LTD., Yongin 17066, Republic of Korea, ⁴Bio-Integration Research Center for Neutra-Pharmaceutical Epigenetics, Chung-Ang University, Seoul 06974, Republic of Korea¹Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 06974, Republic of Korea, ²Aquatic Disease Control Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea, ³Green Cross Veterinary Products Co. LTD., Yongin 17066, Republic of Korea, ⁴Bio-Integration Research Center for Neutra-Pharmaceutical Epigenetics, Chung-Ang University, Seoul 06974, Republic of Korea

Corresponding author: Hyun Ah Kang , Tel: +82-2-820-5863,

Received: 29 May 2017 • Revised: 4 July 2017 • Accepted: 4 July 2017

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Abstract

Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARSbased plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNVCP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.

Keywords: Yarrowia lipolytica;capsid proteins;multicopy integration;nervous necrosis virus;ribosomal DNA;viruslike particles

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Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus

J. Microbiol. 2017;55(8):655-664. Published online July 28, 2017

DOI: https://doi.org/10.1007/s12275-017-7218-5

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